Leveraging deep survival models to predict quality of care risk in diverse hospital readmissions.

Hospital readmissions rate is reportedly high and has caused huge financial burden on health care systems in many countries. It is viewed as an important indicator of health care providers' quality of care. We examine the use of machine learning-based survival analysis to assess quality of care risk in hospital readmissions. This study applies various survival models to explore the risk of hospital readmissions given patient demographics and their respective hospital discharges extracted from a health care claims dataset. We explore advanced feature representation techniques such as BioBERT and Node2Vec to encode high-dimensional diagnosis code features. To our knowledge, this study is the first to apply deep-learning based survival-analysis models for predicting hospital readmission risk agnostic of specific medical conditions and a fixed window for readmission. We found that modeling the time from discharge date to readmission date as a Weibull distribution as in the SparseDeepWeiSurv model yields the best discriminative power and calibration. In addition, embedding representations of the diagnosis codes do not contribute to improvement in model performance. We find dependency of each model's performance on the time point at which it is evaluated. This time dependency of the models' performance on the health care claims data may necessitate a different choice of model in quality of care issue detection at different points in time. We show the effectiveness of deep-learning based survival-analysis models in estimating the quality of care risk in hospital readmissions.

Efficacy and safety of gluten peptide-based antigen-specific immunotherapy (Nexvax2) in adults with coeliac disease after bolus exposure to gluten (RESET CeD): an interim analysis of a terminated randomised, double-blind, placebo-controlled phase 2 study.

BACKGROUND: A gluten-free diet is insufficient to treat coeliac disease because intestinal injury persists and acute reactions with cytokine release follow gluten exposure. Nexvax2 is a specific immunotherapy using immunodominant peptides recognised by gluten-specific CD4+ T cells that might modify gluten-induced disease in coeliac disease. We aimed to assess the effects of Nexvax2 on gluten-induced symptoms and immune activation in patients with coeliac disease. METHODS: This was a randomised, double-blind, placebo-controlled phase 2 trial done at 41 sites (29 community, one secondary, and 11 tertiary centres) in the USA, Australia, and New Zealand. Patients with coeliac disease aged 18-70 years who had excluded gluten for at least 1 year, were HLA-DQ2.5 positive, and had a worsening of symptoms after an unmasked 10 g vital gluten challenge were eligible for inclusion. Patients were stratified by HLA-DQ2.5 status (HLA-DQ2.5 non-homozygous vs homozygous). Patients who were non-homozygous were centrally (ICON; Dublin, Ireland) randomly assigned (1:1) to receive subcutaneous Nexvax2 (non-homozygous Nexvax2 group) or saline (0·9% sodium chloride; non-homozygous placebo group) twice a week escalating from 1 µg to 750 µg during the first 5 weeks followed by 11 weeks of maintenance therapy at 900 µg per dose. The exploratory homozygous group was centrally randomly assigned (2:1) to receive Nexvax2 (homozygous Nexvax2 group) or placebo (homozygous placebo group); patients who were homozygous received the same dosage as those who were non-homozygous. The primary endpoint was change in coeliac disease patient reported outcomes (total gastrointestinal domain) from pretreatment baseline to the day of masked bolus 10 g vital gluten challenge given in week 14 analysed in the non-homozygous intention-to-treat population. The trial is registered with ClinicalTrials.gov, NCT03644069. FINDINGS: Between Sept 21, 2018, and April 24, 2019, 383 volunteers were screened for inclusion, of whom 179 (47%; 133 [74%] women, 46 [26%] men; median age 41 years [IQR 33-55]) were randomly assigned. One (1%) of 179 patients was excluded from analysis due to misassignment of genotype. The non-homozygous Nexvax2 group included 76 patients, the non-homozygous placebo group included 78 patients, the homozygous Nexvax2 group included 16 patients, and the homozygous placebo group included eight patients. The study was discontinued after planned interim analysis of 66 patients who were non-homozygous. We report an unmasked post-hoc analysis of all available data for the primary endpoint and secondary symptom-based endpoints combining data from 67 (66 were assessed in the planned interim analysis for the primary endpoint). Mean change from baseline to day of first masked gluten challenge in total gastrointestinal score for the non-homozygous Nexvax2 group was 2·86 (SD 2·28) compared with 2·63 (2·07) for the non-homozygous placebo group (p=0·43). Adverse events were similar between all patients who received Nexvax2 and those who received placebo. Serious adverse events were reported in five (3%) of 178 patients (two [2%] of 92 who received Nexvax2 and three [4%] of 82 who received placebo). One patient in the non-homozygous Nexvax2 group had a serious adverse event that occurred during gluten challenge (left-sided mid-back muscle strain with imaging suggestive of partial left kidney infarction). Serious adverse events were reported for three (4%) of 78 patients in the non-homozygous placebo group (one each with exacerbation of asthma and appendicitis, and one who had forehead abscess, conjunctivitis, and folliculitis) and one (1%) patient in the non-homozygous Nexvax2 group developed a pulmonary embolism. The most frequent adverse events in all 92 patients who received Nexvax2 compared with all 86 patients who received placebo were nausea (44 [48%] of 92 patients who received Nexvax2 vs 29 (34%) of 86 patients who received placebo), diarrhoea (32 [35%] vs 25 [29%]), abdominal pain (31 [34%] vs 27 [31%]), headache 32 [35%] vs 20 [23%]), and fatigue (24 [26%] vs 31 [36%]). INTERPRETATION: Nexvax2 did not reduce acute gluten-induced symptoms. Masked bolus vital gluten challenge provides an alternative to extended gluten challenge in efficacy studies for coeliac disease. FUNDING: ImmusanT.

Deep immune phenotyping reveals similarities between aging, Down syndrome, and autoimmunity.

Individuals with Down syndrome show cellular and clinical features of dysregulated aging of the immune system, including a shift from naïve to memory T cells and increased incidence of autoimmunity. However, a quantitative understanding of how various immune compartments change with age in Down syndrome remains lacking. Here, we performed deep immunophenotyping of a cohort of individuals with Down syndrome across the life span, selecting for autoimmunity-free individuals. We simultaneously interrogated age- and sex-matched healthy controls and people with type 1 diabetes as a representative autoimmune disease. We built an analytical software, IMPACD (Iterative Machine-assisted Permutational Analysis of Cytometry Data), that enabled us to rapidly identify many features of immune dysregulation in Down syndrome shared with other autoimmune diseases. We found quantitative and qualitative dysregulation of naïve CD4+ and CD8+ T cells in individuals with Down syndrome and identified interleukin-6 as a candidate driver of some of these changes, thus extending the consideration of immunopathologic cytokines in Down syndrome beyond interferons. We used immune cellular composition to generate three linear models of aging (immune clocks) trained on control participants. All three immune clocks demonstrated advanced immune aging in individuals with Down syndrome. One of these clocks, informed by Down syndrome­relevant biology, also showed advanced immune aging in individuals with type 1 diabetes. Orthologous RNA sequencing­derived immune clocks also demonstrated advanced immune aging in individuals with Down syndrome. Together, our findings demonstrate an approach to studying immune aging in Down syndrome that may have implications in other autoimmune diseases.

A Sensitive Whole Blood Assay Detects Antigen-Stimulated Cytokine Release From CD4+ T Cells and Facilitates Immunomonitoring in a Phase 2 Clinical Trial of Nexvax2 in Coeliac Disease.

Improved blood tests assessing the functional status of rare gluten-specific CD4+ T cells are needed to effectively monitor experimental therapies for coeliac disease (CD). Our aim was to develop a simple, but highly sensitive cytokine release assay (CRA) for gluten-specific CD4+ T cells that did not require patients to undergo a prior gluten challenge, and would be practical in large, multi-centre clinical trials. We developed an enhanced CRA and used it in a phase 2 clinical trial ("RESET CeD") of Nexvax2, a peptide-based immunotherapy for CD. Two participants with treated CD were assessed in a pilot study prior to and six days after a 3-day gluten challenge. Dye-dilution proliferation in peripheral blood mononuclear cells (PBMC) was assessed, and IL-2, IFN-γ and IL-10 were measured by multiplex electrochemiluminescence immunoassay (ECL) after 24-hour gluten-peptide stimulation of whole blood or matched PBMC. Subsequently, gluten-specific CD4+ T cells in blood were assessed in a subgroup of the RESET CeD Study participants who received Nexvax2 (maintenance dose 900 µg, n = 12) or placebo (n = 9). The pilot study showed that gluten peptides induced IL-2, IFN-γ and IL-10 release from PBMCs attributable to CD4+ T cells, but the PBMC CRA was substantially less sensitive than whole blood CRA. Only modest gluten peptide-stimulated IL-2 release could be detected without prior gluten challenge using PBMC. In contrast, whole blood CRA enabled detection of IL-2 and IFN-γ before and after gluten challenge. IL-2 and IFN-γ release in whole blood required more than 6 hours incubation. Delay in whole blood incubation of more than three hours from collection substantially reduced antigen-stimulated IL-2 and IFN-γ secretion. Nexvax2, but not placebo treatment in the RESET CeD Study was associated with significant reductions in gluten peptide-stimulated whole blood IL-2 and IFN-γ release, and CD4+ T cell proliferation. We conclude that using fresh whole blood instead of PBMC substantially enhances cytokine secretion stimulated by gluten peptides, and enables assessment of rare gluten-specific CD4+ T cells without requiring CD patients to undertake a gluten challenge. Whole blood assessment coupled with ultra-sensitive cytokine detection shows promise in the monitoring of rare antigen-specific T cells in clinical studies.

Patient factors influencing acute gluten reactions and cytokine release in treated coeliac disease.

BACKGROUND: Patients with coeliac disease (CD) commonly report a variety of adverse symptoms to gluten, but descriptions of the symptomatic response in the literature may have been confounded by the presence of food components such as fermentable carbohydrates (FODMAPs) causing symptoms of irritable bowel syndrome independent of gluten. In recent unmasked and masked low FODMAP gluten challenge studies in small groups of treated CD patients, nausea and vomiting were shown to be the key symptoms associated with serum interleukin (IL)-2 release. Our objective was to utilise a large and diverse cohort of people with CD undertaking a standardised gluten food challenge to characterise the demographic, genetic and clinical factors influencing the severity and timing of acute gluten reactions and IL-2 production. METHODS: A total of 295 adults treated for CD were observed for 6 h after an unmasked food challenge consisting of 10 g vital wheat gluten (low in FODMAPs) in 100 ml water. Assessments included patient-reported outcomes, serum IL-2 and adverse events. Responses were analysed according to patient characteristics, HLA-DQ genotype, duodenal histology and response to a second gluten challenge. RESULTS: Peak symptom severity was at 3 h (median severity 5/10). Peak IL-2 was at 4 h (median 4 pg/ml, range undetectable to 1028 pg/ml). Older age, older age at diagnosis, HLA-DQ2.5 positivity and homozygosity for HLA-DQB1*02 were each significantly associated with IL-2 elevations after gluten. Patients positive for HLA-DQ2.5, DQ8, DQ2.2 or DQ7 showed elevated IL-2 after gluten. Patient factors were not significantly associated with severity of digestive symptoms, but symptoms were correlated to one another and serum IL-2. Gluten challenge after 5 months caused more vomiting and higher IL-2 levels, but responses correlated with the first. CONCLUSIONS: Gluten-induced symptoms and cytokine release is common in adults with treated CD. Age, genetics and previous response to gluten predict these acute reactions to gluten challenge. Structured symptom assessment and serum IL-2 after standardised gluten challenge may inform on patient diagnosis, the role of gluten in symptomatology and the need for adjunctive treatment. TRIAL REGISTRATION: ClinicalTrials.gov , NCT03644069 Registered 21 May 2018.

Cytokine release after gluten ingestion differentiates coeliac disease from self-reported gluten sensitivity.

BACKGROUND: Diagnosing coeliac disease (CD) in patients on a gluten-free diet (GFD) is difficult. Ingesting gluten elevates circulating interleukin (IL)-2, IL-8 and IL-10 in CD patients on a GFD. OBJECTIVE: We tested whether cytokine release after gluten ingestion differentiates patients with CD from those with self-reported gluten sensitivity (SR-GS). METHODS: Australian patients with CD (n = 26) and SR-GS (n = 18) on a GFD consumed bread (estimated gluten 6 g). Serum at baseline and at 3 and 4 h was tested for IL-2, IL-8 and IL-10. Separately, Norwegian SR-GS patients (n = 49) had plasma cytokine assessment at baseline and at 2, 4 and 6 h after food bars containing gluten (5.7 g), fructan or placebo in a previous double-blind crossover study. RESULTS: Gluten significantly elevated serum IL-2, IL-8 and IL-10 at 3 and 4 h in patients with CD but not SR-GS. The highest median fold-change from baseline at 4 h was for IL-2 (8.06, IQR: 1.52-24.0; P < 0.0001, Wilcoxon test). The two SR-GS cohorts included only one (1.5%) confirmed IL-2 responder, and cytokine responses to fructan and placebo were no different to gluten. Overall, cytokine release after gluten was present in 22 (85%) CD participants, but 2 of the 4 non-responders remained clinically well after 1 y on an unrestricted diet. Hence, cytokine release occurred in 22 (92%) of 24 'verified' CD participants. CONCLUSIONS: Gluten challenge with high-sensitivity cytokine assessment differentiates CD from SR-GS in patients on a GFD and identifies patients likely to tolerate gluten reintroduction. Systemic cytokine release indicating early immune activation by gluten in CD individuals cannot be detected in SR-GS individuals.

Masked bolus gluten challenge low in FODMAPs implicates nausea and vomiting as key symptoms associated with immune activation in treated coeliac disease.

BACKGROUND: In patients with coeliac disease, FODMAPs in gluten-containing foods, and participant anticipation of a harmful ('nocebo') effect, may contribute to acute symptoms after gluten challenge. AIM: To establish acute gluten-specific symptoms linked to immune activation in coeliac disease METHODS: We included 36 coeliac disease patients on a gluten-free diet receiving placebo in the RESET CeD trial. Double-blind, bolus vital wheat gluten (~6-g gluten protein) and sham challenges low in FODMAPs were consumed 2 weeks apart. Assessments included daily Coeliac Disease Patient Reported Outcome (CeD PRO) symptom scores (0-10), adverse events and serum interleukin-2 (baseline and 4 hours). RESULTS: Median CeD PRO score for nausea increased most (sham: 0 vs gluten: 5.5; P < .001). Apart from tiredness (1 vs 4, P = .005) and headache (0 vs 2, P = .002), changes in other symptoms were small or absent. Only nausea increased significantly in occurrence with gluten (11% vs 69%, P < .001). Without nausea, only tiredness and flatulence were common after gluten. Nausea (6% vs 61%, P < .001; median onset: 1:34 hours) and vomiting (0% vs 44%, P < .001; 1:51 hours) were the only adverse events more common with gluten than sham. Interleukin-2 was always below the level of quantitation (0.5 pg/mL) at baseline, and after sham. Interleukin-2 was elevated after gluten in 97% of patients (median fold-change: 20), and correlated with severity of nausea (rs  = .49, P = .0025) and occurrence of vomiting (P = .0005). CONCLUSIONS: Nausea and vomiting are relatively specific indicators of acute gluten ingestion, and correlate with immune activation. IBS-like symptoms without nausea are unlikely to indicate recent gluten exposure.

Elevated serum interleukin-2 after gluten correlates with symptoms and is a potential diagnostic biomarker for coeliac disease.

BACKGROUND: Coeliac disease patients on a gluten-free diet experience reactions to gluten, but these are not well characterised or understood. Systemic cytokine release was recently linked to reactivation of gluten immunity in coeliac disease. AIM: To define the nature and time-course of symptoms and interleukin-2 changes specific for coeliac disease patients. METHODS: 25 coeliac disease patients on a gluten-free diet and 25 healthy volunteers consumed a standardised 6 gram gluten challenge. Coeliac Disease Patient-Reported Outcome survey and global digestive symptom assessment were completed hourly up to 6 hours after gluten. Adverse events over 48 hours were recorded. Serum interleukin-2 was measured at baseline, and 2, 4 and 6 hours. RESULTS: Serum interleukin-2 was always undetectable in healthy controls, whereas it was undetectable at baseline and elevated >0.5 pg/ml at 4 hours in 92% of coeliac disease patients. All patient-reported outcome severity scores increased significantly after gluten in coeliac disease patients (P < .001 Wilcoxon signed rank test), but not in controls. Symptoms began after 1 hour, and peaked in the third. Nausea and vomiting characterised severe reactions, but mild reactions were limited to headache and tiredness. Peak interleukin-2 correlated with symptom severity, particularly for nausea and vomiting. CONCLUSIONS: Serum interleukin-2 elevations correlate with timing and severity of symptoms after gluten in coeliac disease. Standardised bolus gluten food challenge and interleukin-2 assessment could provide a valuable clinical test to monitor and diagnose coeliac disease in patients established on a gluten-free diet.

Cytokine release and gastrointestinal symptoms after gluten challenge in celiac disease.

Celiac disease (CeD), caused by immune reactions to cereal gluten, is treated with gluten -elimination diets. Within hours of gluten exposure, either perorally or extraorally by intradermal injection, treated patients experience gastrointestinal symptoms. To test whether gluten exposure leads to systemic cytokine production time -related to symptoms, series of multiplex cytokine measurements were obtained in CeD patients after gluten challenge. Peptide injection elevated at least 15 plasma cytokines, with IL-2, IL-8, and IL-10 being most prominent (fold-change increase at 4 hours of 272, 11, and 1.2, respectively). IL-2 and IL-8 were the only cytokines elevated at 2 hours, preceding onset of symptoms. After gluten ingestion, IL-2 was the earliest and most prominent cytokine (15-fold change at 4 hours). Supported by studies of patient-derived gluten-specific T cell clones and primary lymphocytes, our observations indicate that gluten-specific CD4+ T cells are rapidly reactivated by antigen -exposure likely causing CeD-associated gastrointestinal symptoms.

Randomised clinical trial: a placebo-controlled study of subcutaneous or intradermal NEXVAX2, an investigational immunomodulatory peptide therapy for coeliac disease.

BACKGROUND: Nexvax2 contains three gluten-derived peptides, intended to tolerize coeliac disease patients to gluten. Sequences cover six epitopes that trigger immune activation in human leucocyte antigen-DQ2.5-positive patients, most notably after an initial dose. Patients experience gastrointestinal symptoms with increases in serum interleukin-2. Consistent with Nexvax2's induction of non-responsiveness, reactivity disappears after repeated doses, or is avoided with gradual dose escalation. Early clinical trials used intradermal dosing, but pharmacokinetics and rapid onset of effect suggest that subcutaneous delivery may also be effective. AIMS: To document the relative bioavailability of Nevax2 peptides after subcutaneous and intradermal dosing, and the tolerability and ability of subcutaneous dosing to induce non-responsiveness to Nexvax2 peptides. METHODS: A randomised, double-blind, placebo-controlled study was conducted to assess plasma pharmacokinetics after subcutaneous and intradermal Nexvax2 dosing in HLA DQ2.5-positive patients, who had symptoms after an oral gluten challenge. Randomisation was to semi-weekly Nexvax2 (n = 12) or placebo (n = 2) injections, over a 5-week subcutaneous dose escalation and 2-week maintenance period, the latter with four doses of 900 µg, two subcutaneous and two intradermal. Post-dose circulating peptide and interleukin-2 levels were assessed. Investigators recorded adverse events experienced by patients. RESULTS: Subcutaneous dosing resulted in slightly greater exposure. Interleukin-2 responses were seen with the gluten challenge but not after subcutaneous or intradermal dosing of 900 µg. Adverse events were generally mild and self-limited. CONCLUSIONS: Subcutaneous and intradermal dosing of Nexvax2 yield similar bioavailability of constituent peptides; subcutaneous dose escalation avoids an immune response to dominant gluten epitopes.

Nitric Oxide Engages an Anti-inflammatory Feedback Loop Mediated by Peroxiredoxin 5 in Phagocytes.

Phagocyte microbiocidal mechanisms and inflammatory cytokine production are temporally coordinated, although their respective interdependencies remain incompletely understood. Here, we identify a nitric-oxide-mediated antioxidant response as a negative feedback regulator of inflammatory cytokine production in phagocytes. In this context, Keap1 functions as a cellular redox sensor that responds to elevated reactive nitrogen intermediates by eliciting an adaptive transcriptional program controlled by Nrf2 and comprised of antioxidant genes, including Prdx5. We demonstrate that engaging the antioxidant response is sufficient to suppress Toll-like receptor (TLR)-induced cytokine production in dendritic cells and that Prdx5 is required for attenuation of inflammatory cytokine production. Collectively, these findings delineate the reciprocal regulation of inflammation and cellular redox systems in myeloid cells.

Does long term colchicine prevent degradation of collagen fiber network in osteoarthritis?

AIM: Several studies have shown that colchicine exhibits an improvement in the symptoms of knee osteoarthritis (OA) but its effect on disease progression is unknown. To clarify the mechanism of action this study was done to see if colchicine prevents degradation of collagen fiber network in OA by studying serum cartilage oligomeric matrix protein (COMP) levels, a marker of cartilage turnover, over a period of 1 year. METHODS: Seventy-five patients received colchicine plus paracetamol and 75 received placebo alone for the same time period. Serum COMP assays were done at baseline, 2 months and 1 year by enzyme-linked immunosorbent assay. These markers were compared between visits using repeated measures analysis of variance. RESULTS: Serum COMP levels in the paracetamol-alone group did not show significant change between baseline and 2 months; however, there was a significant increase in serum COMP levels from 2 months to 1 year, suggesting increased uncoupling of proteoglycans from collagen and disease progression. No such change was seen in the colchicine group, signifying lack of progression of disease in this group. CONCLUSION: Colchicine may act as a disease-modifying agent in OA.

Epitope-specific immunotherapy targeting CD4-positive T cells in coeliac disease: two randomised, double-blind, placebo-controlled phase 1 studies.

BACKGROUND: A gluten-free diet is the only means to manage coeliac disease, a permanent immune intolerance to gluten. We developed a therapeutic vaccine, Nexvax2, designed to treat coeliac disease. Nexvax2 is an adjuvant-free mix of three peptides that include immunodominant epitopes for gluten-specific CD4-positive T cells. The vaccine is intended to engage and render gluten-specific CD4-positive T cells unresponsive to further antigenic stimulation. We assessed the safety and pharmacodynamics of the vaccine in patients with coeliac disease on a gluten-free diet. METHODS: We did two randomised, double-blind, placebo-controlled, phase 1 studies at 12 community sites in Australia, New Zealand, and the USA, in HLA-DQ2·5-positive patients aged 18-70 years who had coeliac disease and were on a gluten-free diet. In the screening period for ascending dose cohorts, participants were randomly assigned (1:1) by central randomisation with a simple block method to a double-blind crossover, placebo-controlled oral gluten challenge. Participants with a negative interferon γ release assay to Nexvax2 peptides after the screening oral gluten challenge were discontinued before dosing. For the biopsy cohorts, the screening period included an endoscopy, and participants with duodenal histology who had a Marsh score of greater than 1 were discontinued before dosing. Participants were subsequently randomly assigned to either Nexvax2 or placebo in ascending dose cohorts (2:1) and in biopsy cohorts (1:1) by central randomisation with a simple block method. In the three-dose study, participants received either Nexvax2 60 µg, 90 µg, or 150 µg weekly, or placebo over 15 days; in a fourth biopsy cohort, patients received either Nexvax2 at the maximum tolerated dose (MTD) or placebo. In the 16-dose study, participants received Nexvax2 150 µg or 300 µg or placebo twice weekly over 53 days; in a third biopsy cohort, patients also received either Nexvax2 at the MTD or placebo. In the 4-week post-treatment period, ascending dose cohorts underwent a further double-blind crossover, placebo-controlled oral gluten challenge, which had a fixed sequence, and biopsy cohorts had a gastroscopy with duodenal biopsies and quantitative histology within 2 weeks without oral gluten challenge. Participants, investigators, and study staff were masked to the treatment assignment, except for the study pharmacist. The primary endpoint was the number and percentage of adverse events in the treatment period in an intention-to-treat analysis. Both trials were completed and closed before data analysis. Trials were registered with the Australian New Zealand Clinical Trials Registry, numbers ACTRN12612000355875 and ACTRN12613001331729. FINDINGS: Participants were enrolled from Nov 28, 2012, to Aug 14, 2014, in the three-dose study, and from Aug 3, 2012, to Sept 10, 2013, in the 16-dose study. Overall, 62 (57%) of 108 participants were randomly assigned after oral gluten challenge and 20 (71%) of 28 participants were randomly assigned after endoscopy. In the three-dose study, nine participants were randomly allocated to Nexvax2 60 µg and three to placebo (first cohort), nine were allocated to Nexvax2 90 µg and four to placebo (second cohort), eight were allocated to Nexvax2 150 µg and four to placebo (third cohort), and three were allocated to Nexvax2 150 µg and three to placebo (biopsy cohort). In the 16-dose study, eight participants were randomly allocated to Nexvax2 150 µg and four to placebo (first cohort), ten were allocated to Nexvax2 300 µg and three to placebo (second cohort), and seven were allocated to Nexvax2 150 µg and seven to placebo (biopsy cohort). The MTD for Nexvax2 was 150 µg because of transient, acute gastrointestinal adverse events with onset 2-5 h after initial doses of the vaccine, similar to those caused by gluten ingestion. In the ascending dose cohorts in the three-dose study, six (55%) of 11 placebo recipients, five (56%) of nine who received Nexvax2 60 µg, seven (78%) of nine who received Nexvax2 90 µg, and five (63%) of eight who received Nexvax2 150 µg had at least one treatment-emergent adverse event, as did all three (100%) placebo recipients and one (33%) of three Nexvax2 150 µg recipients in the biopsy cohort. In the ascending dose cohorts of the 16-dose study, five (71%) of seven placebo-treated participants, six (75%) of eight who received Nexvax2 150 µg, and all ten (100%) who received Nexvax2 300 µg had at least one treatment-emergent adverse event, as did six (86%) of seven placebo recipients and five (71%) of seven Nexvax2 150 µg recipients in the biopsy cohort. Vomiting, nausea, and headache were the only treatment-emergent adverse events that occurred in at least 5% of participants in either study. Among participants given the MTD, eight gastrointestinal treatment-emergent adverse events occurred in four (50%) of eight participants in the third cohort and none (0%) of three participants in the biopsy cohort in the three-dose study, and five events occurred in five (63%) of eight participants in the first cohort and three events in two (29%) of seven participants in the biopsy cohort of the 16-dose study. Median villous height to crypt depth ratio in distal duodenal biopsies was not significantly different between those who received the vaccine at the MTD on either schedule and those who received placebo. Of the participants who completed the post-treatment oral gluten challenge per protocol, interferon γ release assay to Nexvax2 peptides was negative (responders to treatment) in two (22%) of nine placebo-treated participants in the three-dose study versus two (33%) of six who received Nexvax2 60 µg, five (63%) of eight who received Nexvax2 90 µg, and six (100%) of six who received Nexvax2 150 µg (p=0·007); in the 16-dose study, none (0%) of five placebo-treated participants had a negative assay versus six (75%) of eight who received Nexvax2 150 µg (p=0·021). INTERPRETATION: The MTD of Nexvax2 was 150 µg for twice weekly intradermal administration over 8 weeks, which modified immune responsiveness to Nexvax2 peptides without deterioration in duodenal histology. The gastrointestinal symptoms that followed the first intradermal administration of the vaccine resembled those associated with oral gluten challenge. These findings support continued clinical development of this potential therapeutic vaccine for coeliac disease. FUNDING: ImmusanT.

Correction: Heterogeneity in Comparisons of Discontinuation of Tumor Necrosis Factor Antagonists in Rheumatoid Arthritis-A Meta-Analysis.

[This corrects the article DOI: 10.1371/journal.pone.0168005.].

Drug Misuse in Adolescents Presenting to the Emergency Department.

OBJECTIVES: Drug misuse is a disturbing, common practice among youth. One in 4 American adolescents reports consuming prescription medications without a clinical indication. We sought to explore current trends of drug misuse in adolescents. METHODS: Using the 37 participating sites of the ToxIC (Toxicology Investigators Consortium) Case Registry, a cross-country surveillance tool, we conducted an observational cohort study of all adolescents (aged 13-18 years) who presented to emergency departments with drug misuse and required a bedside medical toxicology consultation between January 2010 and June 2013. RESULTS: Of 3043 poisonings, 202 (7%) involved drug misuse (139 [69%] were males). Illicit drugs (primarily synthetic cannabinoids and "bath salts") were encountered in 101 (50%), followed by prescription medications (56 [28%]) and over-the-counter (OTC) drugs (51 [25%]). Dextromethorphan was the most commonly misused legal medication (24 [12%]). Polypharmacy exposure was documented in 74 (37%). One hundred sixty-three adolescents (81%) were symptomatic; of these, 81% had central nervous system impairments: psychosis (38%), agitation (30%), coma (26%), myoclonus (11%), and seizures (10%); and 66 (41%) displayed a specific toxidrome, most commonly sedative-hypnotic. Benzodiazepines were the most frequently administered medications (46%). Antidotes were administered to 28% of adolescents, primarily naloxone, physostigmine, N-acetyl-cysteine, and flumazenil. No deaths were recorded. CONCLUSIONS: Adolescents presenting with drug misuse may be exposed to a wide range and combinations of therapeutics or illicit substances and frequently display central nervous system abnormalities, compromising the ability to obtain a reliable history. Frontline clinicians should maintain a high index of suspicion, as routine toxicology screenings fail to detect most contemporary misused legal and designer drugs.

Heterogeneity in Comparisons of Discontinuation of Tumor Necrosis Factor Antagonists in Rheumatoid Arthritis - A Meta-Analysis.

OBJECTIVE: We did a systematic review of studies comparing discontinuation of tumor necrosis factor alpha (TNF) antagonists in rheumatoid arthritis (RA) patients, pooled hazard ratios and assessed clinical and methodological heterogeneity. METHODS: We searched MEDLINE and EMBASE until June 2015 for pairwise hazard ratios for discontinuing infliximab, etanercept, and adalimumab from cohorts of RA patients. Hazard ratios were pooled using inverse variance weighting and random effects estimates of the combined hazard ratio were obtained. Clinical and methodological heterogeneity was assessed using the between-subgroup I-square statistics and meta-regression. RESULTS: Twenty-four unique studies were eligible and large heterogeneity (I-square statistics > 50%) was observed in all comparisons. Type of data, location, and order of treatment (first or second line) modified the magnitude and direction of discontinuation comparing infliximab with either adalimumab or etanercept; however, some heterogeneity remained. No effect modifier was identified when adalimumab and etanercept were compared. CONCLUSION: Heterogeneity in studies comparing discontinuation of TNF antagonists in RA is partially explained by type of data, location, and order of treatment. Pooling hazard ratios for discontinuing TNF antagonists is inappropriate because largely unexplained heterogeneity was demonstrated when random effect estimates were calculated.

RNF166 Determines Recruitment of Adaptor Proteins during Antibacterial Autophagy.

Xenophagy is a form of selective autophagy that involves the targeting and elimination of intracellular pathogens through several recognition, recruitment, and ubiquitination events. E3 ubiquitin ligases control substrate selectivity in the ubiquitination cascade; however, systematic approaches to map the role of E3 ligases in antibacterial autophagy have been lacking. We screened more than 600 putative human E3 ligases, identifying E3 ligases that are required for adaptor protein recruitment and LC3-bacteria colocalization, critical steps in antibacterial autophagy. An unbiased informatics approach pinpointed RNF166 as a key gene that interacts with the autophagy network and controls the recruitment of ubiquitin as well as the autophagy adaptors p62 and NDP52 to bacteria. Mechanistic studies demonstrated that RNF166 catalyzes K29- and K33-linked polyubiquitination of p62 at residues K91 and K189. Thus, our study expands the catalog of E3 ligases that mediate antibacterial autophagy and identifies a critical role for RNF166 in this process.

Characterization of candidate genes in inflammatory bowel disease-associated risk loci.

GWAS have linked SNPs to risk of inflammatory bowel disease (IBD), but a systematic characterization of disease-associated genes has been lacking. Prior studies utilized microarrays that did not capture many genes encoded within risk loci or defined expression quantitative trait loci (eQTLs) using peripheral blood, which is not the target tissue in IBD. To address these gaps, we sought to characterize the expression of IBD-associated risk genes in disease-relevant tissues and in the setting of active IBD. Terminal ileal (TI) and colonic mucosal tissues were obtained from patients with Crohn's disease or ulcerative colitis and from healthy controls. We developed a NanoString code set to profile 678 genes within IBD risk loci. A subset of patients and controls were genotyped for IBD-associated risk SNPs. Analyses included differential expression and variance analysis, weighted gene coexpression network analysis, and eQTL analysis. We identified 116 genes that discriminate between healthy TI and colon samples and uncovered patterns in variance of gene expression that highlight heterogeneity of disease. We identified 107 coexpressed gene pairs for which transcriptional regulation is either conserved or reversed in an inflammation-independent or -dependent manner. We demonstrate that on average approximately 60% of disease-associated genes are differentially expressed in inflamed tissue. Last, we identified eQTLs with either genotype-only effects on expression or an interaction effect between genotype and inflammation. Our data reinforce tissue specificity of expression in disease-associated candidate genes, highlight genes and gene pairs that are regulated in disease-relevant tissue and inflammation, and provide a foundation to advance the understanding of IBD pathogenesis.

Genetic Coding Variant in GPR65 Alters Lysosomal pH and Links Lysosomal Dysfunction with Colitis Risk.

Although numerous polymorphisms have been associated with inflammatory bowel disease (IBD), identifying the function of these genetic factors has proved challenging. Here we identified a role for nine genes in IBD susceptibility loci in antibacterial autophagy and characterized a role for one of these genes, GPR65, in maintaining lysosome function. Mice lacking Gpr65, a proton-sensing G protein-coupled receptor, showed increased susceptibly to bacteria-induced colitis. Epithelial cells and macrophages lacking GPR65 exhibited impaired clearance of intracellular bacteria and accumulation of aberrant lysosomes. Similarly, IBD patient cells and epithelial cells expressing an IBD-associated missense variant, GPR65 I231L, displayed aberrant lysosomal pH resulting in lysosomal dysfunction, impaired bacterial restriction, and altered lipid droplet formation. The GPR65 I231L polymorphism was sufficient to confer decreased GPR65 signaling. Collectively, these data establish a role for GPR65 in IBD susceptibility and identify lysosomal dysfunction as a potentially causative element in IBD pathogenesis with effects on cellular homeostasis and defense.